Cytosolic threonine aldolase is the main soruce of glycine for heme synthesis.

<p>A) Glycine can be synthesized in glucose grown yeast via three enzymes, Gly1 (threonine aldolase), and cytosolic (Shm2) and mitochondrial (Shm1) serine hydroxymethyltransferases. B) Yeast cells of the indicated genotypes were grown to mid-log phase and cells were processed for glycine and heme determination. Wild type glycine was 6.0 nmol/10⁸ cells. C) Cells of the indicated genotypes were grown to mid-log phase and 1:10 serial dilutions plated on SD medium with no supplements.</p

Hem25 is required for the effective import of glycine into mitochondria.

<p>A) Glycine can serve as the sole nitrogen source in <i>S</i>. <i>cerevisiae</i>. The GCV converts glycine to NH₃, as the GCV resides in the mitochondria the use of glycine as a nitrogen source requires efficient uptake of glycine into the mitochondria. B) Inactivation of the <i>HEM25</i> gene in yeast substantially decreased their ability to grow on glycine as the sole nitrogen source. Cells were grown in SD medium containing 30 g/l glycine as nitrogen source. Growth was determined by optical density (OD) of the culture at 600 nm. Data shown are the mean ± SEM for four replicates for wild type and <i>lpd1</i>Δ cells and nine replicates for <i>hem25</i>Δ cells. C) Serine is synthesized from the glycolytic intermediate 3-phosphoglycerate through a series of reactions that includes phosphoserine transaminase (PSAT1 in humans, Ser1 in <i>S</i>. <i>cerevisiae</i>). Serine is normally the main source of one carbon units (CH₂-THF, 5,10 methylenetetrahydrofolate and its metabolites) in cells. Inactivation of the <i>SER1</i> gene in yeast results in yeast cells that are auxotrophic for serine. Glycine supplementation can also overcome a mutation in the <i>SER1</i> gene as glycine can serve as a metabolic source for both serine and one carbon units. However, this capacity depends entirely on mitochondrial glycine import. The import of glycine into the mitochondria can generate one carbon units in the form of CH₂-THF through the activity of the glycine cleavage system (GCV). In addition, mitochondrial serine hydroxymethyltransferase (SHMT2 in humans, Shm1 in yeast) catalyzes the synthesis of serine from glycine and CH₂-THF, with serine exported into the cytoplasm to be consumed for several anabolic pathways including the synthesis of CH₂-THF. Simultaneously, CH₂-THF generated from glycine is oxidized to formate and also exported into the cytoplasm as a source of cytoplasmic one carbon units. D) An inability to import glycine into the mitochondria prevents glycine supplementation from providing serine and one carbon units to cells with an inactivated <i>SER1</i> gene. This was found to be the case upon inactivation of the yeast <i>HEM25</i> gene. Cells were grown to mid-log phase in SD medium containing 1 mM serine, and 1:10 serial dilutions plated on SD medium with no supplements or supplemented with serine or glycine.</p

Identification of a putative second mitochondrial glycine importer.

<p>Yeast cells of the indicated genotypes were grown to mid-log phase in the presence of no supplements or the addition of 5 mM glycine or 50 μg/ml 5-Ala. Cells were processed for heme determination.</p

Rescue of the zebrafish model of congenital sideroblastic anemia.

<p>A) Whole-mount <i>in situ</i> hybridizations with probes for <i>slc25a38a</i> and <i>slc25a38b</i> at 24 and 34 hpf stages of development demonstrate predominant erythroid expression. For each stage and probe, head and tail views are shown and sites of expression are labeled. Abbreviations: pbi–posterior blood island, pcv–posterior cardinal vein, ss–somites, bl–blood, ret–retina, br–brain, h–heart. Expression of <i>slc25a38b</i> was observed in the posterior blood island, blood and posterior cardinal vein consistent with its preferential expression in erythroid progenitors and erythrocytes, whereas <i>slc25a38a</i> had the same blood-related expression pattern features and was also expressed in retina, brain and somites at 24 hpf. B) Representative images for hemoglobin staining using <i>o</i>-dianisidine of 48 hpf zebrafish embryos injected with <i>slc25a38a+b</i> morpholinos, or standard control morpholino (STD MO), treated with 100 mM glycine, 1 mM sodium folate, or both, starting from 4 hours post-injection. To control for morpholino specificity, we also injected 5-mismatch (5MM) versions of the same <i>slc25a38a+b</i> morpholinos and stained the injected embryos with o-dianisidine. For STD MO and 5MM morphants, images from only the STD MO untreated group are presented because of the low variation in <i>o</i>-dianisidine scores of untreated embryos versus those where glycine and/or folate were present. C) Graph of the <i>o</i>-dianisidine staining heme scores of 48 hpf zebrafish embryos injected with either <i>slc25a38a+b</i> morpholinos (slcMO) or STD MO and then treated from 4 through to 48 hpf with glycine, folate, or both glycine plus folate. All embryos were scored as having “low”, “medium” or “normal” hemoglobin levels in a blinded manner based on visual inspection of <i>o</i>-dianisidine staining. The total numbers (right axis) of scored embryos are indicated, from a total of three independent experiments. The “***” indicates that the <i>p</i>-value between the numbers of embryos in different scoring categories for <i>slc25a38a+b</i> morphants treated with glycine and folate versus the untreated <i>slc25a38a+b</i> morphant group is <0.001.</p

Rescue of growth and heme content in yeast models of congentical sideroblastic anemia.

<p>A) Yeast cells were grown to mid-log phase in the absence or presence of 5 mM glycine, 1 mM serine or 50 μg/ml 5-Ala (the <i>hem1</i>Δ strain is normally grown in the presence of 0.5 μg/ml 5-Ala to allow for growth at a wild type rate) and cells were processed for heme determination. Heme values are the mean ± SEM of at least six independent determinations. B) Yeast cells of the indicated genotypes were grown to mid-log phase and processed for heme determination. C) Cells were grown to mid-log phase in SD medium containing 1 mM serine, and 1:10 serial dilutions plated on SD medium supplemented with glycine, 5-Ala, or serine. D) Cells were grown to mid-log phase in the absence or presence of 5 mM glycine, 1 mM serine or 50 μg/ml 5-Ala and processed for heme determination.</p

Pedigree of the Maritime Canadian family.

<p>Clinically affected individuals are indicated with shaded symbols. Individuals for whom DNA samples were collected are indicated by a number. Individuals shaded within 2 quarter sections were affected but were negative for the MAP3K6 mutation. Individuals shaded on one half had another, non-gastric, cancer. Generations I–VI are indicated.</p

Regions with LOD>0.5 from parametric linkage analyses using Merlin when individual 2447 is treated as unknown (sub-pedigree).

<p>Genomic intervals and associated LOD scores are shown under dominant models with 50% or 99% penetrance. Regions are defined by their 1-LOD support interval. Base pair positions are from hg19. An asterisk indicates the SNP was the first or last analyzed marker on the chromosome.</p><p>Regions with LOD>0.5 from parametric linkage analyses using Merlin when individual 2447 is treated as unknown (sub-pedigree).</p

Summary information for each of the germline and somatic mutations found in <i>MAP3K6</i>.

a<p>The H506Y mutation is a somatic second-hit observed in FFPE tumor tissue from patient 1884.</p><p>Summary information for each of the germline and somatic mutations found in <i>MAP3K6</i>.</p

Regions with LOD>1 from pedigree-wide parametric linkage analyses using Merlin.

<p>Genomic intervals and associated LOD scores are shown under dominant models with 50% or 99% penetrance. Regions are defined by their 1-LOD support interval. Base pair positions are from hg19. An asterisk indicates the SNP was the first or last analyzed marker on the chromosome.</p><p>Regions with LOD>1 from pedigree-wide parametric linkage analyses using Merlin.</p

A) Pedigree of the Portuguese Familial Gastric Cancer family.

<p>Affected individuals are shaded in black with the sequenced proband indicated with a triangle. Deceased individuals are marked with a strike-through. Generations I–III are indicated. <b>B</b>) Tumor cells showing signet ring cell morphology (H&E, 200×). <b>C</b>) Tumor cells retaining E-cadherin protein expression (IHC analysis performed with the rabbit anti-E-cadherin Antibody (24E10 Cell Signaling, MA, USA), according to manufacturer's instructions, 200×).</p